This invention relates to the field of detection of opioid receptor sites in normal and abnormal tissue cells and subcellular particles and, more particularly, to novel methods for detection and quantification of opioid receptors in such substrates, and novel fluorescent non-peptide opioid agonists and antagonists useful in such methods.
In the study of various neurological disorders, addictions, tolerances, and other pathological conditions of the brain, efforts have been made to develop correlations between such conditions and the histochemical characteristics of brain tissue containing opioid receptor sites. Most of what is known about opioid/receptor interaction has evolved from studies using radiolabeled lignds. These have proved useful for studies at the level of whole animals, organs, tissues, single cells and subcellular fractions.
Fournie-Zaluski et al., Biochem. Biophys. Res. Comm., Vol. 83, pp. 300-305 (1978), and Hazum et al., Science, Vol. 206, pp. 1077-1079 (1979), describe fluorescent enkephalin derivatives having biological activity. These fluorescent peptide reagents have been used for a fluorometric study of the degradation of enkephalins by aminopeptidases from mouse striatum, Guyon et al., Biochem. Biophys. Res. Comm., Vol. 88, pp. 919-926 (1979), and for the study of opioid clustering of enkephalin receptors in neuroblastoma cells, Hazum et al. supra, and Hazum et al., Nature, Vol. 282, pp. 626-628 (1979). Attempts have also been made in the art to develop fluorescent labeled non-peptide opioids. For example, Ullman U.S. Pat. No. 4,160,016 describes a competitive binding assay for determining various ligands by incubating with a ligand analog-fluorescer and an anti-ligand which binds with both the unknown and the ligand analog. Ullman U.S. Pat. No. 4,161,515 uses the same type of ligand analog-fluorescer in a competitive binding assay for the determination of anti-ligand. Among the ligand analog-fluorescers disclosed by Ullman are morphine and morphine derivatives having a fluorescer bound to the 3 position of the opioid.
Correa et al. "Fluorescent Probes of .alpha. and .beta. Adrenergic and Opiate Receptors: Biochemical and Histochemical Evaluation" Neurosci. Lett., Vol. 16, pp. 47-53 (1980), describe a fluorescent derivative of naloxone prepared by condensing a dansyl derivative at the 6-position of naloxazone. This fluorescent reagent was tested as a potential in vivo fluorescent label for opioid receptors but was found not to be practical since lipofuscin, which is endogenous to the brain tissue, exhibited autofluorescence at the same frequencies at which fluorescent emissions were obtainable from the dansyl moiety. However, by radiolabeling it was determined that the dansyl substituted nalaxozone had an affinity for opioid receptor sites, a result which the authors deemed surprising in view of the relatively large size of the dansyl moiety.